The burden of hepatitis C virus (HCV) infection worldwide is very large, with most of the personal and economic burden yet to come, as cirrhosis and cancer take years to develop. Most infected individuals do not clear the disease, but develop chronic infections that often lead to end-stage liver disease. Current treatment is limited to co-treatment with ribavirin, interferon ? and telaprevir, a therapy that is expensive and not effective in all patients. This R37 renewal application will study important HCV RNA-protein interactions that regulate HCV gene amplification. A highly-structured RNA element is located in the viral 5' noncoding region that functions as an internal ribosome entry site (IRES) that can directly recruit 40S ribosomal subunits to the viral genome. Specifically, we will employ biochemical, NMR and single-molecule fluorescence approaches to explore the timing and control of translation on IRES-initiated mRNAs. In particular, we will study the essential role of ribosomal protein RPS25 in IRES-mediated translation. Secondly, we will study the interaction of RNA helicase RIG-I with viral and host RNA ligands. Specifically, the polymerization dynamics of RIG-I on viral RNA will be studies by atomic force spectroscopy in vitro and ultra-resolution light microcospy in infected cells. Third, the functional roles for the binding sites in the viral noncoding regions for RNA binding protein PGBP2 will be examined by mutagenesis and SHAPE analysis in infected cells. Finally, a role for 5' end-bound microRNA-122 in the protection of the viral RNA from 5' to 3' exonuclease XRN2 will be examined in infected cells. Structural, dynamic and mechanistic analysis of HCV non-coding region structures and interacting RNA binding proteins that modulate HCV gene expression will provide new antiviral targets. RELEVANCE (See instructions): There are no effective antiviral reagents against hepatitis C virus. The 5' end of the viral genome contains an unusual internal ribosome entry site that modulates viral gene expression. Thus, studying the structure and function of this RNA element with viral and cellular proteins will point to novel antiviral intervention.